The present invention relates to a method for measuring the concentration of a specific component contained in a sample to be detected, and a measuring apparatus thereof. More particularly, the present invention relates to a method for measuring the concentration of protein, and the concentration of glucose in a urine collected from a human, or other animals.
The glucose concentration in a urine (i.e., urine sugar value) and the protein concentration in a urine (i.e., urine protein value) reflect a part of the health condition. Then, there has been a demand for an easy and accurate measuring method thereof.
A conventional urinalysis has been accomplished in the following manner. That is, a test paper impregnated with a reagent corresponding to each inspection item such as sugar or protein is dipped in a urine. Then, the color reaction of the test paper is observed by means of a spectrophotometer or the like. With this method, a different test paper is required for each inspection item, and a new test paper is required for every inspection. Therefore, there has occurred a problem of a high running cost. Further, there has also been a limit as to the automation of a urinalysis for laborsaving.
Especially when such test papers are used at home, an amateur is required to perform setting and exchanging of the test papers. This operation is relatively complicated, and disliked, thus inhibiting an urinalysis apparatus from coming into widespread use at home.
In contrast, in PCT International Publication No. 97/18470, there is proposed a method of urinalysis requiring no consumable items such as test papers. This method is based on the notice that glucose and albumin exhibit optical activities, while the other urine components exhibit almost no optical activities. Namely, with this method of urinalysis, the urine sugar value and the urine protein value are determined by measuring the angle of rotation of the urine.
When a light is propagated in a liquid containing an optical active substance, the polarization direction of the light rotates in proportion to the concentration of the optical active substance. That is, the formula (1):A=L×α  (1)where L denotes a measured optical path length, A denotes an angle of rotation (degree), and a denotes a specific rotatory power is satisfied.
For example, when a light with a wavelength of 589 nm is propagated 100 mm in an aqueous glucose solution with a concentration of 100 mg/dl, the polarization direction of the light rotates 50×10−3 degrees. By utilizing such characteristics, it is possible to determine the urine sugar value and the urine protein value from the formula (1). Herein, the respective specific rotatory powers of glucose and albumin at 20° C. are shown in Table 1.
TABLE 1Wavelength (nm)589670Specific rotatory power (degree)Glucose5040Albumin−60−40
When N types of optical active substances are contained in the liquid, the formula (1) is reexpressed as the following formula (2):A=L×(α1×C1+α2×C2+ . . . +αN×CN)  (2)where L denotes a measured optical path length, A denotes an angle of rotation (degree), and αN denotes the specific rotatory power of a substance “n”, N is a natural number of from 1 to n, and CN denotes the concentration (kg/l) of the substance “n”.
As apparent from the formula (2), the information on a plurality of optical active substance concentrations are included in the angle of rotation of the liquid obtained by measurement. Namely, the sum of the angle of rotation attributed to glucose and the angle of rotation attributed to albumin is included in the angle of rotation obtained for a urine.
From the fact that the specific rotatory power varies according to the wavelength of a light to be propagated, by using lights with a plurality of wavelengths, their respective specific rotatory powers are measured, thereby making it possible to determine the urine sugar value and the urine protein value from simultaneous equations comprising a plurality of the equations (2).
With this method, when one type of light source is used, if one of the urine sugar value and the urine protein value is known, it is possible to calculate the other value. However, when both the urine sugar value and the urine protein value are unknown, there occurs a problem that a plurality of light sources are required.
Further, with a conventional method for measuring the concentration of a solution, when the angle of rotation of a sample to be detected containing protein such as a urine or the like is measured, the sample has been required to be heated up to a relatively high temperature in the case where the sample is required to be opacified by heating.
The opacification phenomenon is affected by the pH of the sample to be detected. For example, the temperature at which opacification starts increases when the sample to be detected becomes alkaline. Namely, the opacification starting temperature increases with an increase in pH.
Therefore, in the case of a urine with a high pH, the urine is not opacified unless it is heated up to around 100° C. Since the urine can be heated up to only about 100° C. under an ordinary pressure, the urine may not be opacified when it is strongly alkaline. This is remarkable especially when the protein concentration is low.
Further, if the heating temperature exceeds about 80° C. for opacifying the urine with a high pH, a metal salt and the like suddenly tend to adhere to the walls of a sample cell, or the like. For this reason, there occurs a problem that the upkeep cost for removing them is increased. At the same time, for heating the urine up to around 100° C., i.e., the boiling point, it becomes necessary to reduce temperature distribution (nonuniformity) by heating to improve the temperature control accuracy so that the bumping or the like is avoided, resulting in more rigorous requirements for the apparatus performances. On the other hand, the heating rate is also required to be restricted for reducing the inconsistency in heating, resulting in a longer measurement time. Herein, it is noted that the “temperatuere distribution” means that the temperature of a solution to be detected is varied part by part. In other words, the temperature of the solution is not uniform and has a temperature variation part by part.
Further, when the sample to be detected is a urine, phosphate, carbonate, and the like have high concentrations, and hence the sample may become turbid by precipitation thereof from before heating. Upon heating such a urine, which has already been turbid from before heating, the opacification due to protein is further mixed therewith to affect the dynamic range of the urine protein concentration measurement.
It is therefore an object of the present invention to solve the foregoing problems. Namely, it is an object of the present invention to provide a method of urinalysis in which by heating and opacifying a sample to be detected (solution to be detected) containing protein such as a urine, projecting a light on the sample, and measuring the intensity of the light transmitted through the sample or the light scattered from the sample, the protein concentration of the sample can be evaluated with high precision.